This invention relates to the synthesis, deprotection, and purification of RNA.
Generally, RNA molecules are chemically synthesized and purified by methodologies based on the use of tetrazole to activate the RNA phosphoramidite, ethanolic-NH.sub.4 OH to remove the exocyclic amino protecting groups, tetra-n-butylammonium fluoride (TBAF) to remove the 2'-OH alkylsilyl protecting groups, and gel purification and analysis of the deprotected RNA. Examples of chemical synthesis, deprotection, purification and analysis procedures for RNA are provided by Usman et al., 1987 J Am. Chem. Soc., 109, 7845; Scaringe et al. Nucleic Acids Res. 1990, 18, 5433-5341; Perreault et al. Biochemistry 1991, 30 4020-4025; Slim and Gait Nucleic Acids Res. 1991, 19, 1183-1188. All the above noted references are all hereby incorporated by reference herein.
The deprotection process commonly involves the deprotection of the exocyclic amino protecting groups by NH.sub.4 OH, which is time consuming (6-24 h) and inefficient. This step is then followed by treatment with TBAF to facilitate the removal of alkylsilyl protecting groups, which again is time consuming and not very effective in achieving efficient deprotection.
A recent modification of this two-step strategy for oligoribonucleotide deprotection has been reported by Wincott et al., (Nucleic Acids Res., 1995, 23, 2677-2784) and by Vinayak et al, (Nucleic Acids Symposium series, 1995. 33, 123-125). The optimized conditions make use of aqueous methylamnine at 65.degree. C. for 15 minutes in place of the ammonium hydroxide cocktail to remove exocyclic amino protecting groups while the desilylation treatment needed to remove the 2'-OH alkylsilyl protecting groups utilizes a mixture of triethylamine trihydrogen fluoride (TEA.3HF), N-methyl-pyrrolidinone and triethylamine at 65.degree. C. for 90 minutes, thereby replacing tetrabutyl ammonium fluoride.
Stinchcomb et al., International PCT Publication No. WO 95/23225 describe a process for one pot deprotection of RNA. On page 73, it states that:
"In an attempt to minimize the time required for deprotection and to simplify the process of deprotection of RNA synthesized on a large scale, applicant describes a one pot deprotection protocol . . . According to this protocol, anhydrous methylamine is used in place of aqueous methyl amine. Base deprotection is carried out at 65.degree. C. for 15 minutes and the reaction is allowed to cool for 10 min. Deprotection of 2'-hydroxyl groups is then carried out in the same container for 90 minutes in a TEA.3HF reagent. The reaction is quenched with 16 mM TEAB solution."